Acinetobacter venetianus RAG-1 has been studied for its ability to metabolize a variety of carbon sources such as long chain hydrocarbons, alcohols, fatty acids, and triglycerides and to generate an exopolymer termed emulsan (Gorkovenko, A.; Zhang, J.; Gross, R. A.; Kaplan, D. L. Carbohydrate Polymers 1999, 1, 79-84). The bacterium has been reported to secrete this anionic lipoheteropolysaccharide known as emulsan to aid in the capture and transport of the carbon sources to the cell (Pines, O.; Bayer, E. A.; Gutnick, D. L. Journal of Bacteriology 1983, 2, 893-905). Emulsan has been described as a 1 MDa anionic lipopolysaccharide composed of the three amino sugars, D-galactosamine, D-galactosamineuronic acid, and diamino-6-deoxy-D-glucose, present in a ratio of 1:1:1 (Zuckerberg, A.; Diver, A.; Peeri, Z. Applied and Environmental Microbiology 1979, 3, 414-420). Saturated and monounsaturated fatty acids ranging from C10-C18 were reported to be linked to the polysaccharide backbone by O- and N-acyl bonds and to constitute up to 23% w/w of the polymer (Belsky, I.; Gutnick, D. L.; Rosenberg, E. FEBS Letters 1979, 1, 175-178). According to the previous literature, the emulsan amino groups are either acylated or covalently linked by an amide bond to 3-hydroxybutyric acid (Panilaitis, B.; Johri, A.; Blank, W.; Kaplan, D.; Fuhrman, J. Clinical and Diagnostic Laboratory Immunology 2002, 6, 1240-1247). The combination of hydrophilic anionic sugar main chain repeat units and the hydrophobic side groups were believed to impart an amphipathic behavior to emulsan and result in its ability to form stable oil-in-water emulsions (Rosenberg, E.; Zuckerberg, A.; Rubinovitz, C.; Gutnick, D. L. Applied and Environmental Microbiology 1979, 3, 402-408). Traditionally, studies with emulsan have focused on environmental applications, such as biodegradable surfactants, crude oil viscosity modifiers and the removal of heavy metals (Gutnick, D. L.; Bach, H. Applied Microbiology and Biotechnology 2000, 4, 451-460; Pines, O.; Gutnick, D. Applied and Environmental Microbiology 1986, 3, 661-663; Zosim, Z.; Gutnick, D.; Rosenberg, E. Biotechnology and Bioengineering 1983, 7, 1725-1735).
Recently, emulsan has been investigated for biological uses such as a vaccine adjuvant and as a drug delivery vehicle (Panilaitis, B.; Johri, A.; Blank, W.; Kaplan, D.; Fuhrman, J. Clinical and Diagnostic Laboratory Immunology 2002, 6, 1240-1247; Castro, G. R.; Kamdar, R. R.; Panilaitis, B.; Kaplan, D. L. Journal of Controlled Release 2005, 1-3, 149-157; Castro, G. R.; Panilaitis, B.; Bora, E.; Kaplan, D. L. Molecular Pharmaceutics 2007, 1, 33-46). However, in order to utilize emulsan in these types of biomedical applications, an improved purification process was required to avoid potential contaminants with undesirable biological side effects. Contaminants such as lipopolysaccharides (LPS), host cell protein (HCP), nucleic acids (DNA), and residual salts and solvents can potentially exhibit toxic effects on mammalian systems if present even in relatively low concentrations (Garnick, R. L.; Ross, M. J.; du Mee, Charles P. In Encyclopedia of Pharmaceutical Technology; Swarbick, J., Boyland, J. C., Eds.; Marcel Dekker: New York, N.Y., 1988; Vol. 1, pp 253-313). The purification of emulsan, first published in 1979, has traditionally been conducted using four steps; cell removal by centrifugation, ammonium sulfate precipitation of the polymer, hot phenol extraction to remove contaminating proteins, and dialysis against water to remove the phenol (Zuckerberg, A.; Diver, A.; Peeri, Z. Applied and Environmental Microbiology 1979, 3, 414-420). The scientific literature utilizing this purification approach has supported consistently that the emulsan from this purification scheme is an essentially pure product. However, the prior purification approaches do not result in a pure product, as demonstrated herein.